Study On Medium Optimization, Strain Breeding And Genetic Transformation System Of Aurantiochytrium Limacinum

Docosahexaenoic acid (DHA) is a kind of n-3polyunsaturated fatty acids.Dyerberg found that DHA play an important role in cardiovascular disease. Thenscholars found that DHA was importantespecialy for the infants and young children innervous development, eyesight protection, anti-cancer, anti-inflammatory and so on.The DHA content in Aurantiochytrium limacinum reaches about40%of the dryweight of the cell, so A. limacinum is considered as an ideal DHA production speciesand catches the conern of the scholars. In this paper, the research was carried at manyaspects, including optimization of the culture conditions, breeding of good strains,cloning of function gene and transgenic method research, which layed foundation forimprovment of the biomass and DHA content of A.limacinum.In this paper, A.limacinum was used as the original strain, and MnCl24H2O,ZnSO47H2O, CoCl26H2O were used as three factors, to find the effect of traceelements on growth and DHA content. Orthogonal experimental results showed thatbiomass and DHA content had a positive correlation in A.limacinum. Co2+is the mostsignificant factor affecting the biomass and DHA content, followed by the Zn2+andMn2+. Theoretical optimal combination of the trace elements in the orthogonalexperiment is consistent with the optimal group of experiment, asMnCl24H2O:32mg/L; ZnSO47H2O:30mg/L; CoCl26H2O:1.2mg/L. Adding thetrace elements in A.limacinum increased the biomass61.38%, and increased DHAcontent47.85%.Then to get a new A.limacinum strain with high DHA content, A.limacinumOUC168was used as the original strain to be mutated by NTG mutagenesis, andmutant strain A.limacinum OUCB1was obtained byquizalofop selection. For themutant, the biomass and the DHA content reached7.33g·L-1·d-1and18.05%， respectively, which is65.09%and49.92%higher than those of the original strainA.limacinum OUC168, repectively. An orthogonal test was designed, including thestrains, the contents of glucose, sodium glutamate and yeast extract in the medium asthe four factors, and the biomass, DHA content and ACCase activity were measured.Results showed that among the factors, strain affects the biomass, DHA content andACCase activity more significantly. A positive correlation was found among ACCaseactivity and DHA content. At the same time, best culture condition for OUCB1waspresented as: glucose70g/L, sodium glutamate20g/L, yeast extract20g/L,. Themutant strain OUCB1provided potential application value because of its bettercharacteristics.Polyketide synthase (PKS) is a key enzyme in PUFA synthesis of A.limacinum,which plays a crucial role in DHA synthesis pathway. In this paper, full length of pks2gene was cloned, which contains6,105bp nucleotides with no intron. This6,105bpnucleotide sequence encodes a complete protein of2034animo acids, whichcontains four conserved domains, namely KS, AT, and FabD enzyme domain. KSdomain in the amino acid sequence is repeated, but the repeated KS domains are notsame, which may be due to the different substrates of KS. FabD domain which isbelonged to FAS enzyme synthesis pathway was found, and the function of FabDdomain is consistent with the AT domain in PKS biosynthetic pathway. In this paper,full length sequence of pks2was first cloned, which provides a foundation for study ofsynthesis pathway of DHA on the molecular level, and also provides useful molecularinformation in genetic modification on A.limacinum.Finally, Cre/loxP site-specific recombination system is first applied to theA.limacinum. p18SCPGC vector which contains loxP sites, chloramphenicol markerand green fluorescent protein was transferred into A.limacinum OUC168, and arecombinant A.limacinum OUC_EG was sreened, which can grow on mediumcontaining chloramphenicol and has a green fluorescence. Then plasmid pSH65wastransferred into A.limacinum OUC_EG, and Cre recombinase expressed by pSH65removed the chloramphenicol marker gene between the loxP sites. After that, arecombinant A.limacinum OUC_CG which has no chloramphenicol marker gene, but the green fluorescence was still observed. By PCR detection, chloramphenicol markergene in A.limacinum OUC_CG has been verified to be removed successfully. Throughcryogenic fluorescence detection and Southern blotting, the green fluorescent proteinin A.limacinum OUC_EG and OUC_CG were verified not to be infulenced by theexcision of chloramphenicol marker gene, and green fluorescence still remains. Thesuccessful application of Cre/loxP site-specific recombination systems in A.limacinumprovides an experimental basis on genetical modification of A.limacinum andobtainment of transformed strains with no antibiotic marker.